Human cytomegalovirus has been associated with a number of clinical syndromes. The infection is usually asymptomatic and self-limiting in the immunocompetent child or adult, but can cause severe congenital disease in the fetus or infant and morbidity and mortality in immuno-compromised patients. HCMV is a common cause of mental retardation in children who acquire the infection in utero from mothers carrying an active infection, and is considered the leading opportunistic infection causing complications in immunosuppressed transplant patients. HCMV is also an important consideration in the treatment of Acquired Immunodeficiency Syndrome (AIDS). It has recently been suggested that HCMV is the causative agent in Kaposi's sarcoma, a cancer often seen in AIDS.
Much work has been done in recent years on the basic nature of HCMV in order to ameliorate these problems. HCMV is a virus of the herpes type, composed of a nuclear complex of nucleic acid and proteins, internal capsid proteins with structural or enzymatic functions and an external membrane envelope, containing glycopeptides and glycolipids. The first two groups are sequestered within the virion and may not be available in the intact, infectious virion to react with the immune system or antibodies. Furthermore, their presence in bodily fluids is not diagnostic of active infections as it is typical of herpes-type infections to go into a quiescent phase, where viral products are produced but infectious virions are not released. In the same manner, an initial infection may be abortive, i.e., the host cells begin to produce viral products, but these products are not assembled into virions so that active infection is never established.
HCMV can be diagnosed by one or more of the following methods: 1) identification of HCMV in virion bodies or CMV antigens in infected tissues by using indirect immunofluorescent microscopy, 2) detection of the virus in cell monolayers inoculated with infectious tissue or body fluids, 3) identification of HCMV-specific antibody in serum or, 4) hybridization with a complementary-labeled DNA fragment. For a review of current methods of diagnosing HCMV, see Mayo Report (Mayo Clin. Prac., 60, 636, (1985)).
The most common method of diagnosing HCMV has been culturing the sample in human fibroblasts and inspection for typical cytopathic effects. This method has the advantage of being useful with a number of different, easily obtainable samples such as urine, throat washings or blood. After two to four weeks of growth, cultures can be inspected for evidence of HCMV infection, such as inclusion bodies or the typical giant, fused cell. This method has the disadvantage of requiring up to one month before diagnosis is confirmed.
Diagnosis utilizing monoclonal antibodies (MoAbs), using ELISA or direct immunofluorescence has been attempted. For example, Schuster et al. (Mayo Clin. Proc., 60, 577-585 (1985)) have produced MoAbs to early and late HCMV nuclear proteins for use in immunofluorescence. The MoAb can be labeled with a fluorescent material and binding to intracellular HCMV in biopsy specimens or cultured cells can be observed. This kind of diagnosis is much more rapid than the culture method. Results may be available in less than one day.
Emerging antiviral therapies may become useful in treating HCMV infections. In the case of transplant patients, the immunosuppressant drug regimen can be altered to allow reactivation of latent HCMV. Furthermore, the symptoms of HCMV may mimic and therefore mask rejection so that control of transplant rejection requires accurate and rapid diagnosis of possible HCMV-active infections.
Antibodies against viruses can be injected directly into the circulation of HCMV patients, where the antibodies will bind to circulating virions and render them incapable of infecting host cells, thereby directly limiting the infection.
Additionally, nonexposed adults, especially females of childbearing age, may advantageously be administered a vaccine to confer protection from HCMV infection. U.S. Pat. No. 3,959,466 describes such a vaccine, using attenuated HCMV which is passed at least 50 and preferably 125 to 150 times through culture in human lung fibroblasts. Such vaccine is said to induce immunity against HCMV without allowing spread of the virus through the body. However, it is well known in the art that such attenuated viral vaccines, as well as "killed" vaccines, may have a certain low incidence of reversion to a virulent form. This has been well noted in the case of the Salk and Sabin polio vaccines.
Recently, subunit vaccines have been successfully constructed. Such subunit vaccine consists of only a portion of the virus and is not capable of causing the production of active virus. Alternatively, genetic material coding for immunogenic gene products can be spliced to a nonvirulent carrier virus, such as Vaccinia. The recombinant virus has the advantage of being reproduced for a time within the body, therefore stimulating immunity to a greater extent than can a killed virus.
It is becoming increasingly clear that the research on the immunology of HCMV must look to the membrane envelope components, especially the glycopeptides, as immunogens and markers of active HCMV infection. The starting material for a virus preparation includes, in addition to the desired membrane envelope components, host cell debris, nuclear proteins, capsid proteins and precursor peptides which are not found in the mature, infective virion. Even among the membrane envelope glycopeptides, not all are physically accessible to the immune system because they may reside in a fold or crypt or be sterically hindered from interaction with host immune responsive cells.
Therefore, it is desirable to provide a membrane envelope glycopeptide that is physically accessible to interaction with the immune system and with antibodies raised to it, in addition to eliciting antibodies with good in vitro reactivity, e.g., with respect to their binding to the glycopeptide. When such a glycopeptide on the surface of a virion reacts with its corresponding antibody, the virion is rendered incapable of infecting the host cell, that is, it is neutralized.
Such a glycopeptide and antibodies raised against it are useful in diagnosing active HCMV infections, treating such infections and, as a component of combined or subunit vaccines, for preventing infection in susceptible populations.
Before such antibody therapy, diagnosis of active HCMV infection or vaccine can be made available, it is necessary to identify and provide those compositions which stimulate humoral and cellular immunity. The glycopeptides of HCMV and other viruses have proved useful in this respect. For example, detergent extracts of HCMV containing certain membrane envelope glycopeptides can induce humoral and cellular immunity in the guinea pig. MoAbs have been obtained which immunoprecipitate glycopeptides with molecular weights of 50,000-58,000, 93,000 and 130,000 daltons that were associated in disulfide complexes. Some of these antibodies were observed to have neutralizing activity in vitro but only in the presence of complement. Only one MoAb, which reacted with an 86,000 dalton protein of HCMV was neutralizing in the absence of complement (Rasmussen et al., Proc. Natl. Acad. Sci. U.S.A., 81, 876-880 (1984)).
In 1976, Kim et al. (J. Virol., 20, 604-611 (1976)) were able to find at least 33 polypeptides in HCMV, ranging in size from 11,000 to 290,000 daltons. Six of these were shown to be glycopeptides, but the resolution technique, electrophoresis in non-reducing detergent gel, did not resolve these glycopeptides sufficiently to allow characterization of the individual molecules. One viral protein, a glycosylated protein of molecular weight 66,000 was found to be the most abundant viral polypeptide. Kim later succeeded in partially purifying this glycopeptide and raising MoAbs against it. However, none of his MoAbs was capable of neutralizing HCMV even at very high concentration. A mixture of nine MoAbs also failed to exhibit virus-neutralizing activity (Kim et al., J. Clin. Microbiol. 18, 331-343, 1983).
Therefore, a need exists for providing those HCMV glycopeptides capable of eliciting a protective humoral response. An additional needs exists for MoAbs directed against such glycopeptides.